Lipoxygenases are non-heme iron dioxygenase enzymes, which catalyze the hydroperoxidation of dieneoic and polyenoic fatty acids. Lipoxygenases are ubiquitous in mammals and include the 5-, 12-, and 15-lipoxygenases which respectively catalyze the insertion of oxygen at the C-5, C-12 and C-15 positions of arachidonic acid. The resulting leukotrienes and lipoxins provide signaling molecules associated with a variety of human diseases such as asthma, atherosclerosis, psoriasis and inflammatory bowel disease. Selective inhibitors for the human lipoxygenases thus are potentially useful as pharmacological agents, neutraceuticals and/or molecular tools.
Marine natural products have provided a variety of bioactive compounds, but relatively few marine natural products have been noted as being lipoxygenase inhibitors. The pseudopterosins are diterpene glycosides found in the Caribbean sea whip Pseudopterogorgia elisabethae, believed to have anti-inflammatory properties related to inhibition of cycloxygenase and lipoxygenase biosynthetic pathways (Look et al., J. Org. Chem. 1986, 51, 5140; Jacobs et al., Pharm. Lett. 1998, 62, 401). Fuscosides, which are diterpenoid glycosides from marine gorgonia, also have anti-inflammatory properties believed to be associated with lipoxygenase inhibition (Jacobsen et al., J. Pharmacol. Exp. Ther. 1992, 262, 866).
The only sponge-derived lipoxygenase inhibitors to date are pentabromo biphenyl ethers (Fu et al., J. Nat. Prod. 1995, 58, 609) isolated from cyanobacterial-containing sponges. The anti-inflammatory compound manoalide from Luffariella variabilis was initially identified as an inhibitor of 5-lipoxygenase (Devries et al., Biochem. Pharmacol. 1988, 37, 2899), but the available 5-lipoxygenase assay used has been problematic, and subsequent studies (Cabre et al., Inflamm. Res. 1996, 45, 218) show that manoalide has no lipoxygenase inhibitory properties.
There is a need for new selective inhibitors of human lipoxygenases, as well as other anti-inflammatory bioactive compounds usable for treatment of conditions associated with lipoxygenase and other conditions and diseases. The present invention satisfies these needs, as well as others, and overcomes deficiencies found in the background art.
The invention provides new compounds that are effective lipoxygenase inhibitors and effective therapeutic agents for methods and treatments for lipoxygenase mediated conditions and other diseases, disorders and conditions. The invention also provides pharmaceutical compositions and methods for inhibiting human lipoxygenases, treatments for lipoxygenase mediated conditions, and treatments for other diseases, disorders and conditions. The invention further provides pharmaceutical compositions and methods for increasing cell viability and prevention of cell damage associated with ischemia or hypoxic or anoxic events, and for treatment of ischemia, stroke, and inflammatory conditions generally.
The subject compounds, pharmaceutical compositions and methods comprise, or comprise the use of, subersic terpenoids, jaspic terpenoids, igemellic terpenoids, hippospongic terpenoids, halicondric terpenoids, dictyodendric terpenoids, and/or heteronemic terpenoids.
In some embodiments of the invention, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds having the structure: 
wherein R1 comprises 
and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. In specific embodiments, the compounds, pharmaceutical compositions and methods may comprise or utilize (xe2x88x92)-(5R, 10R)-subersic acid, also named 4-hydroxy-3-[3-methyl-5-(2,5,5,8a -tetramethyl-3,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-pent-2-enyl]-benzoic acid (1): 
Substantially purified or isolated subersic terpenoid compounds isolated from Suberea sp. (Order: Verongida, Family: Aplysinellidae) are disclosed, including the novel compound (xe2x88x92)-(5R, 10R)-subersic acid (1), together with methods of isolation and purification thereof. These compounds are shown to be effective inhibitors of lipoxygenases, specifically human 15-lipoxygenase, and are demonstrated to increase cell viability under ischemic and anoxic conditions, and provide effective treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally. Pharmaceutical methods and formulations using (xe2x88x92)-subersic acid (1) and other subersic terpenoids, either derived from natural source such sponge or marine organism, or prepared synthetically, are disclosed. An extract of Suberea sp. (Order: Verongida, Family: Aplysinellidae) is also demonstrated as a lipoxygenase inhibitor.
In other embodiments of the invention, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. In specific embodiments, the subject compounds, pharmaceutical compositions and methods may comprise or utilize the novel compound (+)-(5S, 6S)-subersin, also named 3-[4-methyl-6-(1,2,6-trimethyl-cyclohex-2-enyl)-hex-3-enyl]-furan (2): 
Substantially purified or isolated (+)-(5S, 6S)-subersin (2), together with methods of isolation and purification thereof, are disclosed. (+)-Subersin (2) is shown to be an effective inhibitor of lipoxygenases, specifically human 15-lipoxygenase, is demonstrated to increase cell viability under ischemic and anoxic conditions, and to provide effective treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally. Pharmaceutical methods and formulations using (+)-subersin (2) and other subersic terpenoids, either derived from natural source such sponge or marine organism, or prepared synthetically, are disclosed.
In still other embodiments of the invention, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and wherein the 2,6,6-trimethyl cyclohexenyl moiety is unsaturated at either the 1- or 2- position, as well as individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. In specific embodiments, the subject compounds, pharmaceutical compositions and methods may comprise or utilize jaspaquinol, also named 2-[3,7-dimethyl-9-(2,6,6-trimethyl-cyclohex-1-enyl)-nona-2,6-dienyl]-benzene-1,4-diol (3): 
Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for jaspaquinol (3) and other jaspic terpenoids. These methods and pharmaceutical preparations may comprise the use of one or more jaspic terpenoids derived from Jaspis johnstoni, Jaspis splendens or other Jaspis sp. sponge, or other marine organism or natural source, or which may be synthetically prepared.
In further embodiments of the invention, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. In specific embodiments the compounds, pharmaceutical compositions and methods may comprise or utilize (xe2x88x92) jaspic acid, also named 4-hydroxy-3-[2,5,8a-trimethyl-5-(4-methyl-pent-3-enyl)-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-ylmethyl]-benzoic acid (4): 
Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for jaspic acid (4) and other jaspic terpenoids. These methods and pharmaceutical preparations may comprise the use of one or more jaspic terpenoids derived from Jaspis johnstoni, Jaspis splendens or other Jaspis sp. sponge, or other marine organism or natural source, or which may be synthetically prepared.
In certain embodiments of the invention, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
wherein the 2,6,6-trimethyl cyclohexenyl moiety is unsaturated at either the 1- or 2-position, and wherein R2 comprises: an alkyl group, alkylcarbonyl group, sulfonate, sulfonate salt, or hydrogen, and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. More specifically, the compounds, pharmaceutical compositions and related methods may comprise or utilize hipposulfate C, also named [2-(3-furan-3-yl-propyl)-6-methyl-8-(2,6,6-trimethyl-cyclohex-2-enyl)-oct-5-enyl] sodium sulfate (5), or its corresponding alcohol igernellin (6), also named 2-(3-furan-3-yl-propyl)-6-methyl-8-(2,6,6-trimethyl-cyclohex-2-enyl)-oct-5-en-1-ol: 
wherein R2 comprises: xe2x80x94SO3Na (5) or xe2x80x94H (6). Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for hipposulfate C(R2xe2x95x90SO3Na) (5) and igernellin (R2xe2x95x90H) (6) as well as other igernellic terpenoids and hippospongic terpenoids, which may be synthetically prepared or derived from a natural source such as Igernella sp., Hippospongia sp., Cocinoderma sp or other sponge family such as Halichondriida, or other marine organism.
In some embodiments, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and wherein R2 comprises an alkyl group, alkylcarbonyl group, sulfonate, sulfonate salt, or hydrogen, and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. More specifically, the subject compounds, pharmaceutical compositions and methods may comprise or utilize halisulfate 7 (7), also named {5-furan-3-yl-2-[2-(1,2,5,5-tetramethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-1-yl)-ethyl]- pentyl} sodium sulfate or its corresponding alcohol, 5-furan-3-yl-2-[2-(1,2,5,5-tetramethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-1-yl)-ethyl]-pentan-1-ol (8): 
wherein R2 comprises xe2x80x94SO3Na (7) or xe2x80x94H (8).
Substantially purified or isolated 5-furan-3-yl-2-[2-(1,2,5,5-tetramethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-1-yl)-ethyl]-pentan-1-ol (8), together with methods of isolation and purification thereof, are disclosed. Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for halicondric terpenoids such as Halisulfate 7 (7) and its corresponding alcohol, (8).
In other embodiments, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and wherein R2 comprises: an alkyl group, alkylcarbonyl group, sulfonate, sulfonate salt, or hydrogen, and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. More specifically, the subject compounds, pharmaceutical compositions and related methods may comprise or utilize hipposulfate D (R2xe2x95x90SO3Na) (9), also named 2-[5-hydroxy-4-methyl-6-(2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-hexyl]-2-methyl-2H-chromenyl sodium sulfate, or its corresponding alcohol (R2xe2x95x90H) (10) also named 2-[5-hydroxy-4-methyl-6-(2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a -octahydro-napthalen-1-yl)-hexyl]-2-methyl-2H-chromen-6-ol: 
wherein R2 comprises: xe2x80x94SO3Na (9) or xe2x80x94H (10).
Substantially purified or isolated hipposulfate D (9), together with methods of isolation and purification thereof, are disclosed. Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for hipposulfate D (9), the alcohol (10), and other hippospongic terpenoids.
In still other embodiments, the subject compounds, pharmaceutical compositions and methods may comprise or utilize compounds of the structure: 
wherein R1 comprises: 
and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. More specifically, the subject compounds, pharmaceutical compositions and methods may comprise or utilize dictyodendrillin B, also named 4-(4,8-dimethyl-nona-3,7-dienyl)-5-hydroxy-5H-furan-2-one (11): 
and/or dictyodendrillin C, also named 3-(4,8-dimethyl-nona-3,7-dienyl)-5-hydroxy-5H-furan-2-one (12): 
Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under ischemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for dictyodendrillin B (11), dictyodendrillin C (12), and other dictyodendric terpenoids.
In certain embodiments, the subject compounds, pharmaceutical compositions and related methods may comprise or utilize compounds of the structure: 
wherein each R2 group individually comprises an alkyl group, alkylcarbonyl group, sulfonate, sulfonate salt, or hydrogen, and individual isomers, racemic or non-racemic mixtures of isomers, and pharmaceutically acceptable salts thereof. More specifically, the subject compounds, pharmaceutical compositions and methods may comprise or utilize heteronemin (13), also named 24,25-epoxy-17(24)-scalarene-12-ol-16,25-diacetate 
Methods and pharmaceutical compositions for inhibiting lipoxygenases, treatment of lipoxygenase-mediated conditions, prevention of cell damage and promotion of cell viability under isehemic and/or anoxic conditions in mammals and other subjects, and treatment for ischemia, congestive heart failure, stroke, and inflammatory conditions generally, are shown for heteronemin (13) and other heteronemic terpenoids.
The terpenoid compounds above may be used singly or together in various combinations, as well as in various combinations with other known lipoxygenase inhibitors, other bioactive agents, and pharmaceutically acceptable excipients, to provide pharmaceutical preparations and methods in accordance with the invention. The pharmaceutical methods and compositions of the invention are usable for treatment of a variety of lipoxygenase mediated conditions and inflammatory conditions found in mammals and other subjects. The methods and compositions of the invention are also usable for enhancing cell viability and preventing cell damage associated with ischemia, anoxia or hypoxia in various tissues and organs, and for treatment of ischemia, congestive heart failure, stroke, and inflammatory conditions generally in human and other subjects. The subject compounds are also useful for treatment of ischemia, stroke, congestive heart failure, and inflammation that is not associated with lipoxygenase.
The invention also provides novel compounds in isolated or substantially purified form that are useful for treatment of lipoxygenase-mediated conditions and other diseases, disorders or conditions in humans or other mammals. More particularly, the novel compound (xe2x88x92)-(5R, 10R)-subersic acid, also named 4-hydroxy-3-[3-methyl-5-(2,5,5,8a-tetramethyl-3,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-pent-2-enyl]-benzoic acid (1) is disclosed, together with methods of isolation and purification thereof. 
The novel compound (+)-(5S, 6S)-subersin, also named 3-[4-methyl-6-(1,2,6-trimethyl-cyclohex-2-enyl)-hex-3-enyl]-furan (2) is also disclosed, together with methods of isolation and purification thereof. 
The novel compounds hipposulfate C, also named [2-(3-furan-3-yl-propyl)-6-methyl-8-(2,6,6-trimethyl-cyclohex-2-enyl)-oct-5-enyl] sodium sulfate (5) and its corresponding sulfonic acid are also disclosed, together with methods of isolation and purification thereof. 
The novel compound (10) 2-[5-hydroxy-4-methyl-6-(2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-hexyl]-2-methyl-2H-chromen-6-ol (10) is also disclosed, together with methods of isolation and purification thereof. 
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading of the details more fully described below.
Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
xe2x80x9cSubersic terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Suberea sp. sponge, Cacospongia sp. sponge, or any synthetic equivalents or derivatives thereof. Subersic terpenoids include, by way of example, (xe2x88x92)-subersic acid (1), (+)-subersin (2), jaspaquinol (3) and (xe2x88x92) jaspic acid (4). Certain terpene and terpenoid compounds are found in more than one species of sponge, as will be apparent upon review of the following examples. Thus, the compounds jaspaquinol (3) and (xe2x88x92) jaspic acid (4) are also xe2x80x9cjaspic terpenoidsxe2x80x9d as defined below.
xe2x80x9cJaspic terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Jaspis sp. sponge, or any synthetic equivalents or derivatives thereof. Exemplary jaspic terpenoids include jaspaquinol (3) and (xe2x88x92) jaspic acid (4) (which are also subersic terpenoids as defined above, as jaspaquinol (3) and (xe2x88x92) jaspic acid (4) are isolatable from Suberea sp).
xe2x80x9cIgemellic terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Igernella sp. sponge, or any synthetic equivalents or derivatives thereof. An exemplary igemellic terpenoid is igernellin (6), which is also a hippospongic terpenoid as described below.
xe2x80x9cHippospongic terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Hippospongia sp. sponge, or any synthetic equivalents or derivatives thereof. Exemplary hippospongic terpenoids include hipposulfate C(Rxe2x95x90SO3Na) (5) and its corresponding alcohol igernellin (Rxe2x95x90H) (6), hipposulfate D (Rxe2x95x90SO3Na) (9) and its corresponding alcohol (Rxe2x95x90H) (10), and halisulfate 7 (Rxe2x95x90SO3Na) (7) and its corresponding alcohol (Rxe2x95x90H) (8).
xe2x80x9cHalicondric terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any sponge of the family Halicondriidae, or any synthetic equivalents or derivatives thereof. Exemplary halicondric terpenoids include halisulfates 1-6 (not shown) and halisulfate 7 (7).
xe2x80x9cDictyodendric terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Dictyodendrilla sp., Cacospongia sp. sponge, or any synthetic equivalents or derivatives thereof. Exemplary dictyodendric terpenoids include dictyodendrillin B (11) and dictyodendrillin C (12).
xe2x80x9cHeteronemic terpenoidxe2x80x9d means any terpene or terpenoid compound found in or derived or isolated from any Heteronema sp. sponge, or any synthetic equivalents or derivatives thereof. An exemplary heteronemic terpenoid is heteronemin (13).
A xe2x80x9cpharmaceutically acceptable excipientxe2x80x9d means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. xe2x80x9cA pharmaceutically acceptable excipientxe2x80x9d as used in the specification and claims includes both one and more than one such excipient.
A xe2x80x9cpharmaceutically acceptable saltxe2x80x9d of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4methylbicyclo) 2.2.2!oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4xe2x80x2-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
xe2x80x9cPro-drugsxe2x80x9d means any compound which releases an active parent drug according to formula (I) in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of formula (I) are prepared by modifying functional groups present in the compound of formula (I) in such a way that the modifications may be cleaved in vivo to release the parent compound. Prodrugs include compounds of formula (I) wherein a hydroxy, amino, or sulfhydryl group in compound (I) is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds of formula (I), and the like.
xe2x80x9cTreatingxe2x80x9d or xe2x80x9ctreatmentxe2x80x9d of a condition or disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
A xe2x80x9ctherapeutically effective amountxe2x80x9d means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The xe2x80x9ctherapeutically effective amountxe2x80x9d will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
A xe2x80x9clipoxygenase-mediated conditionxe2x80x9d means any condition, disorder or disease related to or otherwise associated with a lipoxygenase protein or the inhibition thereof, including, by way of example and without limitation, asthma, atherosclerosis, psoriasis and inflammatory bowel disease, anoxia, hypoxia, or anoxia related conditions such as miocardial infarction, cerebral ischemia, retinal ischemia, myocardial ischemia, post surgical cognitive dysfunction and other ischemias, inflammatory diseases and disorders including diabetes, renal disease, pre-menstrual syndrome, asthma, cardiopulmonary inflammatory disorders, heart failure (including chronic and congestive heart failure), rheumatoid arthritis, osteoarthritis, and muscle fatigue, and neurodegenerative disorders including Alzheimer""s, dementia and Parkinson""s disease; peripheral neuropathy including spinal chord injury, head injury and surgical trauma; and allograft tissue and organ transplant rejection, and aging and conditions associated with aging.
xe2x80x9cIschemiaxe2x80x9d means any condition related to or otherwise associated with anoxia, hypoxia or other oxygen-depleted condition, including, for example, local anemia caused by arterial blockage or narrowing in a tissue or organ.
xe2x80x9cLipoxygenasexe2x80x9d means any mammalian or non-mammalian lipoxygenase, including 5-, 12-, and 15-lipoxygenases, and particularly human lipoxygenases.
The terms xe2x80x9csubjectxe2x80x9d and xe2x80x9cpatientxe2x80x9d mean a member or members of any mammalian or non-mammalian species that may have a need for the pharmaceutical methods, compositions and treatments described herein.
xe2x80x9cMammalxe2x80x9d means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, particularly humans. Animal models, particularly small mammals, e.g. murine, lagomorpha, etc. may be used for experimental investigations.
The term xe2x80x9cunit dosage form,xe2x80x9d as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular terpene or terpenoid compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
xe2x80x9cOptionalxe2x80x9d or xe2x80x9coptionallyxe2x80x9d means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, xe2x80x9cheterocyclo group optionally mono- or di-substituted with an alkyl groupxe2x80x9d means that the alkyl may, but need not, be present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted with the alkyl group.
Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed xe2x80x9cisomers.xe2x80x9d Isomers that differ in the arrangement of their atoms in space are termed xe2x80x9cstereoisomers.xe2x80x9d Stereoisomers that are not mirror images of one another are termed xe2x80x9cdiastereomersxe2x80x9d and those that are non-superimposable mirror images of each other are termed xe2x80x9cenantiomers.xe2x80x9d When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (xe2x88x92)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a xe2x80x9cracemic mixture.xe2x80x9d
The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see, e.g., the discussion in Chapter 4 of xe2x80x9cAdvanced Organic Chemistryxe2x80x9d, 4th edition J. March, John Wiley and Sons, New York, 1992).
Before the present compounds and pharmaceutical compositions and methods are described, it is to be understood that this invention is not limited to particular formulations described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms xe2x80x9caxe2x80x9d, xe2x80x9candxe2x80x9d, and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9ca terpenoidxe2x80x9d includes a plurality of such terpenoids, and reference to xe2x80x9ca lipoxygenase inhibitorxe2x80x9d includes reference to one or more lipoxygenase inhibitors and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The invention disclosed herein provides pharmaceutical compositions comprising terpene or terpenoid compounds found in the sponges Suberea sp., Cacospongia sp., Jaspis sp., Igernella sp., Coscinoderma sp., Hippospongia sp., Dictyodendrilla sp., Heteronema sp., or of the family Halichondriidae, and other sponge or natural sources, or synthetic derivatives thereof, which exhibit lipoxygenase inhibition activity or are efficacious in the treatment of lipoxygenase-mediated conditions, or are effective at promoting or improving cell viability or preventing cell damage in various tissues or organs under ischemic, anoxic or hypoxic conditions.
The invention also provides methods for inhibiting lipoxygenase comprising contacting or exposing a lipoxygenase to an effective amount of one or more of the subject compounds. The invention further provides methods for treating a subject suffering from a lipoxygenase-mediated condition comprising administering to the subject a therapeutically effective amount of one or more of the subject compounds.
The invention additionally provides methods for preventing cell damage and/or increasing cell viability in various tissues and organs under ischemic, anoxic or hypoxic conditions comprising contacting or exposing cells to an effective amount of one or more of the subject compounds. The invention further provides methods for treating a subject suffering from ischemia, stroke, inflammatory condition, and anoxia or hypoxia in a tissue or organ, comprising administering to the subject a therapeutically effective amount of one or more of the subject compounds.
By way of example, and not of limitation, the subject compounds may be of synthetic origin, or derived or isolated from the aforementioned sponges, or derived or isolated from other natural source, or prepared synthetically. The subject compounds may comprise, by way of example, one or more compounds selected from the group of: 
wherein R1, comprises: 
and wherein R2 comprises an alkyl group, alkylcarbonyl group, sulfonate, sulfonate salt, or hydrogen.
More specifically, the subject compounds may comprise one or more compounds selected from the group of: 
(xe2x88x92)-(5R, 10R)-subersic acid (1), also named 4-hydroxy-3-[3-methyl-5-(2,5,5,8a-tetramethyl-3,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-pent-2-enyl]-benzoic acid; 
(+)-(5S, 6S)-subersin (2), also named 3-[4-methyl-6-(1,2,6-trimethyl-cyclohex-2-enyl)-hex-3-enyl]-furan; 
jaspaquinol (3), also named 2-[3,7-dimethyl-9-(2,6,6-trimethyl-cyclohex-1-enyl)-nona-2,6-dienyl]-benzene-1,4-diol; 
(xe2x88x92) jaspic acid (4), also named 4-hydroxy-3-[2,5,8a-trimethyl-5-(4-methyl-pent-3-enyl)-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-ylmethyl]-benzoic acid; 
hipposulfate C(R SO3Na) (5), also named [2-(3-furan-3-yl-propyl)-6-methyl-8-(2,6,6-trimethyl-cyclohex-2-enyl)-oct-5-enyl] sodium sulfate or its corresponding alcohol igernellin (Rxe2x95x90H) (6), also named 2-(3-furan-3-yl-propyl)-6-methyl-8-(2,6,6-trimethyl-cyclohex-2-enyl)-oct-5-en-1-ol; 
halisulfate 7 (Rxe2x95x90SO3Na) (7), also named {5-furan-3-yl-2-[2-(1,2,5,5-tetramethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-1-yl)-ethyl]-pentyl} sodium sulfate or its corresponding alcohol (Rxe2x95x90H) (8), also named 5-furan-3-yl-2-[2-(1,2,5,5-tetramethyl-1,2,3,4,4a,5,6,7-octahydro-naphthalen-1-yl)-ethyl]-pentan-1-ol; 
hipposulfate D (Rxe2x95x90SO3Na) (9), also named 2-[5-hydroxy-4-methyl-6-(2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-hexyl]-2-methyl-2H-chromenyl sodium sulfate, or its corresponding alcohol (Rxe2x95x90H) (10), also named 2-[5-hydroxy-4-methyl-6-(2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a-octahydro-naphthalen-1-yl)-hexyl]-2-methyl-2H-chromen-6-ol; 
dictyodendrillin B (11), also named 4-(4,8-dimethyl-nona-3,7-dienyl)-5-hydroxy-5H-furan-2-one; 
dictyodendrillin C (12), also named 3-(4,8-dimethyl-nona-3,7-dienyl)-5-hydroxy-5H-furan-2-one and 
heteronemin (13), also named 24,25-epoxy-17(24)-scalarene-12-ol-16,25-diacetate
The cellular and molecular mechanisms associated with lipoxygenase-mediated conditions, such as those that lead to neuronal damage and inflammation associated with various types of brain ischemia, can be carried out using in vitro model systems, such as primary cell cultures, that retain the metabolic characteristics of neurons in vivo. The use of such cell-based models has led to advances in identification of biochemical mechanisms leading to neuronal death in conditions such as anoxia, hypoglycemia, excitotoxicity, and exposure to reactive oxygen species. Neuronal cell lines such as the pheochromocytoma cell line, PC12, are also useful models for studying the effects of oxidative stress on the structure: and function of neuron-specific proteins that are expressed in the cell lines. As many neuronal cell lines do not express all the properties of genuine neurons, primary neuronal cultures are now widely used as in vitro models in which to discern the processes that occur in intact brain.
In vitro models of ischemia can provide oxygen and glucose deprivation that mimic in vivo conditions, for example, by placing neuronal cultures into large anaerobic or hypoxic chambers and exchanging culture medium with oxygen-free and defined ionic composition media. The toxic overstimulation of neuronal glutamate receptors, especially N-methyl-D-aspartate (NMDA) receptors, contribute to hypoxic-ischemic neuronal injury (Choi, D. M., Neuron 1988, 1: 623-634), ischemic induction of reactive oxygen species (ROS) (Watson, B. D., et al., Ann NY Acad. Sci. 1988, 59: 269-281), excessive calcium influx (Grotta, J. C., Stroke 1988, 19: 447-454), arachidonic acid increase (Siesjo, B. K., J. Cereb. Blood Flow Metab. 1981, 1: 155-186), and DNA damage (MacManus, J. P., et al., Neurosci. Lett., 1993, 164: 89-92) causing a cascade of neurodegeneration.
Primary embryonic hippocampal neuronal cells are widely recognized as models of neuronal function. The hippocampus is a source of a relatively homogenous population of neurons with well-characterized properties typical of central nervous system (CNS) neurons in general. Pyramidal neurons, the principal cell type in the hippocampus, have been estimated to account for 85% to 90% of the total neuronal population (Banker and Goslin, Culturing Nerve Cells, 2nd edition 1998, The MIT Press, Cambridge, Mass.). The hippocampus also exhibits a remarkable capacity for activity-dependent changes in synaptic function, such as long-term potentiation (Hawkins et al, Ann. Rev Neurosci. 1993, 16, 625-665).
In experiments carried out in support of the present invention and shown in the examples below, anoxia/ischemia was induced in primary cultures of hippocampal neuronal cells, and the subject terpenoid compounds of the invention were tested for their ability to prevent cell death due to ischemia. Briefly, primary cultures of hippocampal neurons may be used to test compounds for activity in neuronal protection. Cultures are typically prepared from 18- to 19-day fetal rats. At this age, the generation of pyramidal neurons, which begins in the rat at about E15, is essentially complete. The brain tissue at this stage is relatively easy to dissociate, the meninges are removed readily, and the number of glial cells still is relatively modest (Park et al., J Neurochem 2000, 74(1), 114-124).
In order to determine activity, each compound was assessed for its ability to protect cells against one or more standard stressors, including hypoxia, as detailed in the Examples. Compounds that are neuroprotective in such an in vitro cell systems are usable in appropriate in vivo animal models.
Similarly, primary cultures of myocytes were used to test the subject compounds in vitro for ability to provide protection against heart damage, such as myocardial ischemia or congestive heart failure. Such cells are typically used to study molecular models of myocardial ischemia (Webster et al., J. Mol. Cell Cardiol. 1995, 27, 453-458; Camilleri et al., Cell Biol. and Toxicol. 1997, 13, 435-444; Bielawska et al., Am. J. Pathol. 1997, 151:1257-1263) and therefore can be used to screen for myoprotective compounds, as described herein. Exemplary stressor assays for this purpose are provided in the examples below. For example, cardiomyocytes in culture exhibit contractile (xe2x80x9cbeatingxe2x80x9d) activity; each cardiomyocyte contraction is associated with a rise in intracellular calcium termed a xe2x80x9ccalcium transientxe2x80x9d. These calcium transients can be measured using Fluo-4, a fluorescent dye which exhibits large fluorescence intensity increases on binding calcium. This assay is cell-based and tests the ability of potential cytoprotectant molecules to guard against ischemic damage and allow the cells to maintain their contractile function.
The subject compounds have been further evaluated and validated as pharmacologically efficacious using in vitro assays based on a variety of different cell lines known in the art, as well in vivo via appropriate animal or whole organ assays, such as the isolated heart model of cardiac function, the rat air pouch model for inflammation, the rat middle-cerebral artery occlusion (MCAO) stroke model, and others. Similarly, the subject compounds can be further validated in additional animal models (e.g., diabetes, renal failure, asthma, muscle fatigue, inflammation), such as are well known in the art.
The subject compounds are usable for administration to subjects exhibiting or suffering from lipoxygenase-mediated conditions, diseases or disorders, including any condition, disease or disorder associated with lipoxygenase inhibition and, more specifically in the case of human subjects, with human 15-lipoxygenase inhibition. The subject compounds are usable for administration to subjects exhibiting or suffering from ischemia, hypoxia or anoxia-related conditions. The aforementioned conditions, diseases and disorders include, without limitation, asthma, atherosclerosis, psoriasis and inflammatory bowel disease, anoxia, hypoxia, or anoxia related conditions such as miocardial infarction and cerebral ischemia (stroke), other ischemias, and inflammatory conditions generally. Inflammatory diseases and disorders include without limitation diabetes, renal disease, pre-menstrual syndrome, asthma, cardiopulmonary inflammatory disorders, heart failure (including chronic and congestive heart failure), rheumatoid arthritis, osteoarthritis, and muscle fatigue. The compounds are also usable for administration to subjects exhibiting conditions characterized by oxidative stress including without limitation, ischemia including stroke, congestive heart failure, cerebral ischemia, retinal ischemia, myocardial ischemia, myocardia infarction and post surgical cognitive dysfunction; neurodegenerative disorders including Alzheimer""s, dementia and Parkinson""s disease; peripheral neuropathy including spinal chord injury, head injury and surgical trauma; and for the preservation of allograft tissue or organ for transplantation. Various other diseases, conditions or disorders treatable using the subject compounds, pharmaceutical formulations thereof, and related methods, will suggest themselves to those skilled in the art and are also considered to be within the scope of this disclosure.
Also provided by the invention are pharmaceutical preparations of the subject compounds. The subject compounds can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. The formulations may be designed for administration via a number of different routes, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
In pharmaceutical dosage forms, the subject compounds of the invention may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the subject compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
The subject compounds of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
The compounds of the invention can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the subject compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Depending on the patient and condition being treated and on the administration route, the subject compounds may be administered in dosages of, for example, 0.1 xe2x96xa1g to 10 mg/kg body weight per day. The range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses typically being 20, 30 or even 40 times smaller (per unit body weight) in man than in the rat. Similarly the mode of administration can have a large effect on dosage. Thus, for example, oral dosages may be ten times the injection dose. Higher doses may be used for localized routes of delivery.
A typical dosage may be a solution suitable for intravenous administration; a tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient, etc. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
For use in the subject methods, the subject compounds may be formulated with other pharmaceutically active agents, including other lipoxygenase-inhibiting agents.
Pharmaceutically acceptable excipients usable with the invention, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
Kits with unit doses of the subject compounds, usually in oral or injectable doses, are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating pathological condition of interest. Preferred compounds and unit doses are those described herein above.